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Chromatographia

, Volume 71, Issue 1–2, pp 131–134 | Cite as

LC Determination of Nosiheptide in Swine Kidney and Liver

  • Haiyang Jiang
  • Weihong Zhai
  • Xi Xia
  • Shuangyang Ding
  • Fei Xu
  • Jianzhong Shen
  • Xiaowei Li
  • Jinfeng Liu
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Abstract

A liquid chromatographic method was developed for determination of nosiheptide in swine kidney and liver. The tissue samples were extracted with acetonitrile and defatted with hexane. The analytes were determined at a fluorescence excitation/emission wavelength of 357/515 nm and with gradient elution program. The limits of detection and limits of quantification were 5 and 20 ng g−1, respectively, for swine kidney and liver. The mean recoveries for nosiheptide in swine kidney and liver ranged from 70.3 to 97.4% with coefficients of variation below 11.3% at 20–100 ng g−1 fortification levels.

Keywords

Column liquid chromatography Swine kidney and liver Nosiheptide 

1 Introduction

Nosiheptide is a sulfur-containing polypeptide antibiotic which is commonly used as an animal feed additive for promotion of growth, prevention and control of diseases in farm animals [1, 2]. It was isolated from Streptomyces actuosus 40037 (NRRL 2954) in the 1960s and found to be effective to inhibit bacteria protein synthesis in vivo and in vitro, and not to influence the synthesis of DNA and RNA [3, 4, 5]. The structure of nosiheptide is shown in Fig. 1.
Fig. 1

The structure of nosiheptide

Nosiheptide is quite different from all the other members of this family. It is very active to inhibit infections caused by gram-positive bacteria, with the average maximum inhibitory concentration (MIC) of 8 ng mL−1. But for gram-negative bacteria, such as Escherichia coli, Shigella, Salmonella, Aerobacter, Proteus, Serratia, Pseudomonas, Brucella, Fusobacterium, MIC was more than 125,000 ng mL−1. Moreover, the anti-virus effect of nosiheptide has been reported recently [6, 7, 8, 9].

Due to the significant effect of growth-promoting and disease prevention, nosiheptide is usually administered via feed to animals [10, 11, 12]. However, this may result in problems of residues in edible tissues. To safeguard public health, Japan fixed MRL at 30 ng g−1 for muscle, kidney, liver and fat in the Positive List System for Agriculture Chemical Residues in Food [13].

There are only very limited selective chemical methods reported, because of the absence of chromophores and hydrophilic nature of the polypeptides. The analytical methods mentioned in the literatures include spectrophotometry [14], microbiological assay [15, 16, 17, 18, 19], thin layer chromatography [20, 21], and liquid chromatography (LC) [22, 23, 24]. But so far, there is no report referring to chemical methods of nosiheptide residues in swine kidney and liver.

The object of this paper is to development an effective and sensitive LC method for determination of nosiheptide in swine kidney and liver, and provides a powerful means of test for its monitoring in practice.

2 Experimental

2.1 Materials

Methanol and acetonitrile (chromatographic grade) were purchased from Fisher Scientific (Fairlawn, NJ, USA). Dimethylformamide (DMF), hexane, 1-propanol, sodium tetraborate, and phosphoric acid were obtained from Beijing Chemical Factory, Beijing, PR China. Nosihetide power (purity > 98.5%) was supplied by Zhejiang Esigma Animal Health Co., Ltd., Zhejiang Province, PR China. Demineralized water was obtained by means of a Milli-Q system (Millipore, Bedford, MA, USA).

2.2 Instrumentation and Chromatographic Conditions

The LC system was equipped with a Waters 2475 fluorescence detector and a Waters 2695 separations module (Waters Co., Milford, MA, USA). The chromatographic column was a reversed-phase column (Inertsil ODS-3 columns, 4.6 mm i.d. × 250 mm, 5 μm, GL Sciences Inc. Tokyo, Japan). The injection volume was 20 μL. The column temperature was set at 30 °C. Excitation wavelength was at 357 nm, and emission wavelength was at 515 nm. The mobile phase consisted of solvent A (0.025% phosphoric acid aqueous solution) and solvent B (acetonitrile) and was run at a flow rate of 1.0 mL min−1 with a gradient program as follows: 60% B (0 min), 60–40% B (7 min), 40–100% B (3 min), 100% B (3 min), 100–40% B (1 min), 40% B (11 min).

2.3 Standard and Fortification Solutions

A standard stock solution was prepared by dissolving 0.1 g with DMF and diluting to a final volume of 100 mL with DMF.

The stock solution was diluted with appropriate volumes of methanol to prepare working and fortification solutions. The standard curve for nosiheptide was built in the range of 20–1,000 ng mL−1.

2.4 Fortification

A fortifying test was performed by adding a microvolume of an aqueous standard solution containing nosiheptide to each portion of the weighed samples. The fortification levels for each tissue were 20, 50, and 100 ng g−1. The sample extraction procedure is described below.

2.5 Sample Extraction

The sample extraction for nosiheptide from fortified kidney tissue was performed following a previous report [24]. Briefly, swine kidney tissue was minced and homogenized in a homogenizer for 2 min. Two grams of homogenate was accurately weighed into a polypropylene centrifuge tube. Ten milliliters of acetonitrile were added and vortexed for 1 min. Centrifugation was performed for 10 min at 3,500 rpm (2,300g). The supernatant was removed into a 100 mL polypropylene centrifuge tube, and 50 mL of acetonitrile added to the tissue pellet and the above procedure was repeated. Twenty milliliters of hexane were added to the combined supernatants and shaken for 5 min. The lower acetonitrile layer was transferred into a heart-shape flask and 3 mL of 1-propanol were added to avoid bumping. The mixture was evaporated to dryness on a rotary evaporator under vacuum.

Two grams of liver homogenate were transferred into a 50-mL polypropylene centrifuge tube. One milliliter of 0.1 M sodium tetraborate solution was added and vortexed for 1 min. After standing for 10 min, 10 mL of acetonitrile were added, and the extracts centrifuged for 10 min at 4,000 rpm (3,000g). The clear supernatant was transferred into another tube. The residual sample was extracted with 10 mL acetonitrile and centrifuged again. Twenty milliliters of hexane were added to the combined supernatants and centrifuged for 5 min at 3,000 rpm (1,700g). The upper hexane was removed and 6 g of sodium sulfate were added. After vortexing for 30 s, the mixture was centrifuged for 10 min at 4,000 rpm (3,000g). The acetonitrile layer was transferred into a heart-shape flask and 3 mL of 1-propanol were added to avoid bumping. The mixture was evaporated to dryness on a rotary evaporator under vacuum.

The residuals were dissolved with 2 mL of methanol and passed through a 0.2 μm Nylon membrane syringe filter. 20 μL aliquot of filtrate were injected into the LC system.

2.6 Calculations

The concentration of nosiheptide in tissue samples can be calculated by the following equation:
$$ {\text{Conc}} . ({\text{ng}}\,{\text{g}}^{ - 1} ) = {\frac{(S - B) \times V \times F}{K \times M}} $$
where S = Area of the nosiheptide peak from the chromatogram; B = Intercept of the standard curve; K = Slope of the standard curve; M = Weight of tissue sample; V = Final volume of sample extract; F = Dilution factor.

3 Results

3.1 LC Method Validation

The LC method linearity for nosiheptide was determined over the concentration range of 20–1,000 ng mL−1. The method correlation coefficient was 0.9999. Based on a signal-to-noise ratio of 3:1 and 10:1, the limits of detection and the limits of quantitation, were 5 and 20 ng g−1, respectively, for swine kidney and liver. Method accuracy and precision for the swine tissues were also studied by fortifying the control tissues at the levels of 20, 50, and 100 ng g−1. The recovery data are presented in Table 1. Inter-day mean recoveries of the analytes were 95.3–97.4% and 73.2–78.0%, with coefficients of variation (CVs) of 0.8–9.3% and 5.0–7.7% for swine kidney and liver, respectively. Intra-day mean recoveries of the analytes were 92.3–94.0% and 70.3–72.0%, with CVs of 3.5–10.9% and 8.4–11.3%, respectively. These data met the acceptance criteria for method accuracy, the average recovery was between 70 and 110% and for method precision the CV was 15% or less for each group of samples.
Table 1

Average accuracy and precision of nosiheptide in fortified tissue samples (n = 5)

Samples

Added (ng g−1)

Inter-day

Intra-day

Rec. (%)

CV (%)

Rec. (%)

CV (%)

Kidney

20

96.0

9.3

92.3

10.9

50

97.4

0.8

94.0

5.3

100

95.3

4.4

93.4

3.5

Liver

20

78.0

7.1

70.3

11.3

50

75.6

7.7

71.2

9.9

100

73.2

5.0

72.0

8.4

The typical chromatograms for nosiheptide standard solution and extracts of control and fortified tissues are shown in Fig. 2. The chromatographic conditions using gradient instead of isocratic elution show a good separation without matrix interferences.
Fig. 2

Chromatograms of a nosiheptide standard 20 ng mL−1, b control swine kidney, c fortified swine kidney (20 ng g−1), d control swine liver, e fortified swine liver (20 ng g−1)

4 Conclusions

The described method for the determination of nosiheptide in swine kidney and liver fulfils all acceptance criteria concerning specificity, recovery, linearity, precision and accuracy of calibration and quality control samples.

Notes

Acknowledgment

This work was supported by the Program for New Century Excellent Talents in University (No. NCET-08-0538) and the National Key Technologies Research and Development Program of China during the 11th Five-Year Plan Period (No. 2006BAD31B08).

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Copyright information

© Vieweg+Teubner | GWV Fachverlage GmbH 2009

Authors and Affiliations

  • Haiyang Jiang
    • 1
    • 2
  • Weihong Zhai
    • 3
  • Xi Xia
    • 1
    • 2
  • Shuangyang Ding
    • 1
    • 2
  • Fei Xu
    • 1
  • Jianzhong Shen
    • 1
    • 2
  • Xiaowei Li
    • 1
    • 2
  • Jinfeng Liu
    • 1
    • 2
  1. 1.Department of Pharmacology and Toxicology, College of Veterinary MedicineChina Agricultural UniversityBeijingPeople’s Republic of China
  2. 2.National Reference Laboratory for Veterinary Drug ResidueBeijingPeople’s Republic of China
  3. 3.Qinghai Animal Husbandry & Veterinary CollegeHuangyuanPeople’s Republic of China

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